Effects of cytochalasin B on membrane-associated microfilaments in a cell-free system.

Abstract

The mode of action of cytochalasin B was examined in vitro using bile canaliculusenriched plasma membrane fractions isolated from rat liver. The pericanalicular microfilaments, which are mainly actin filaments and which are normally attached to the canalicular membranes, were dissociated from the membranes by cytochalasin B treatment. A microfilamentous network was found in the supernate of the cytochalasin-treated specimens and a number of polypeptides, of which a polypeptide corresponding in molecular weight to actin was a notable member . These results suggest that actin filaments become detached from the canaliculus membranes by cytochalasin B.The cytochalasins are widely used in biology and pathology as cytopharmacological agents because of their effects on the contractile functions of actin filaments (34) . Alteration of microfilament contractility is a general effect that has been observed in many cell types, but the functional sequelae vary with the specialized nature of the cell involved (4,18,36) . Accompanying these functional changes in cell motility, structural changes have been observed in the electron microscope . The actin filaments (F-actin), which are -5 nm in diameter, disappear and the regions normally occupied by the filaments contain granules and short filaments . It has generally been held that this structural change is a manifestation of disassembled or altered microfilaments (19,30) .Isolated bile canaliculus-enriched plasma membrane fractions of rat liver provide a useful experimental tool for the study of microfilament-membrane association. It has been shown by heavy meromyosin binding (7,24), by immunofluorescent staining, and by immunoelectron microscopic techniques (10, 12) that the pericanalicular microfilaments are mainly actin filaments, and that they remain attached to the canalicular membranes even after the homogenization and centrifugation procedures involved in the isolation of these fractions (6,21) . This model lends itself to an examination of the direct effects of cytochalasin B (CB) on plasma membraneassociated filaments in a cell-free system, and hence gets away from difficulties inherent in the interpretation of results in a more complex cellular system . MATERIALS AND METHODSThe bile canaliculus-enriched fractions of liver cell plasma membranes (BCM) were isolated from the livers of male Wistar rats, weighing -150 g, according to 524 a modification (6) of the method reported by Song et al . (29) . CB (Aldrich Chemical Co . Inc., Milwaukee, Wise .) was dissolved in dimethyl sulfoxide (DMSO) (3 mg of CB/0.1 ml of DMSO) and was diluted with 3 mM phosphatebuffered saline (PBS) (pH 7.4) at an appropriate concentration . Each BCM fraction (2 .2 . mg average protein content) determined by the method of Lowry et al. (l6) was divided into two parts of equal weight . One part was incubated at room temperature for 90 minwith 1.5 ml of PBS containing CB and 0.2% DMSO . Studies were carried out with final CB concentrations of 2.2 x 10-5 M and 2.2 x 10 -6 M. The other pa...

Multi-modal liquid biopsy platform for cancer screening: screening both cancer-associated rare cells and cancer cell-derived vesicles on the fabric filters for a reliable liquid biopsy analysis

“…We first determined the optimal CB concentration and treatment time which could promote cells to release large number of vesicles, while having minimal cytotoxic effect. CB has been well-established as a metabolite which could promote cells to release large number of EVs from their surface by involving in cytoskeleton-membrane interaction [27]. These EVs have been exploited in a wide range of biomedical applications, as they are known to reflect the biological characteristics of the parental cells that they have been originated from [28].…”

Motility of bile canaliculi in the living animal: implications for bile flow.

“…Using isolated hepatocyte couplets and time lapse microscopy, it has been shown that bile canaliculi repeatedly open and close and that this motion is accompanied by the expelling of a bolus of bile (52,55). We have interpreted this motility as active bile canalicular contractions (47,52,54,55,66), but others consider the motile events as noncontrac-tile collapses of canaliculi resulting from secretory pressure with rupture of canaliculi (9,11,30,35). In this report, we have taken advantage of the biliary secretion of sodium fluorescein by hepatocytes to visualize the bile canalicular system and have examined canalicular motility in the living state in order to shed light on these issues.…”

Development of the Bile Canalicular System in Rat Liver: with Special Reference to Bile Canalicular Actin Filaments and Mg2+, Ca2+-ATPase Activity.

“…Bile canalicular F-actin changes its volume in various liver cholestasis. In experimental animals, phalloidin and cytochalasin B treatments make bile canalicular F-actin either overpolymerized or depolymerized, respectively, to cause intracanalicular MV disappearance and intrahepatic jaundice (4,10,19,23). Abnormality of bile canalicular F-actin was frequently observed in patients and animals with obstructive jaundice, drug induced liver injury and other hepatic disorders (9,31).…”